Composition suitable for protecting microorganisms

ABSTRACT

A composition includes at least one carrier containing a polysaccharide, at least one antioxidant and at least one amino acid selected from cysteine, lysine, alanine and arginine. Such a composition can be used for protection of microorganisms during drying, storage and/or reconstitution. A culture powder, a process of making the culture powder, and products containing the culture powder are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 15/580,804 filed on Dec. 8, 2017, which is a National Stage ofInternational Application No. PCT/EP2016/065359 filed on Jun. 30, 2016,which claims priority to European Patent Application No. 15174488.5filed on Jun. 30, 2015, the entire contents of which are beingincorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a composition comprising at least onecarrier comprising a polysaccharide, at least one antioxidant and atleast one amino acid selected from cysteine, lysine, alanine andarginine. It also relates to the use of such a composition for theprotection of microorganisms during drying, storage and/orreconstitution, to a culture powder, to a process of making the culturepowder and to products comprising the culture powder.

BACKGROUND

Beneficial culture powders and in particular probiotic culture powdersare added to a wide range of shelf-stable products. Most low moistureproducts containing probiotic culture powders claim a shelf-life of atleast 18 months. During this period, a minimum amount of live microbesmust be guaranteed in the product. For this reason, the dosage ofculture powder in the product needs to account for the loss in microbialviability during storage. It is therefore important to find ways ofprotecting microorganisms during processing and during storage topromote their survival.

Bacteria experience various stresses during drying under warmconditions, such as heat and shear stress in the dryer or osmotic stressdue to dehydration, for example during spray-drying. These stresses canhave detrimental or even lethal effects on the bacteria. By addition ofprotective agents, the bacteria can be stabilized to a certain degreeagainst these stresses. Further stress is applied on the bacteria duringstorage. Also, when the powder is reconstituted with liquid, thebacteria are exposed to osmotic stress.

Protective agents can stabilize microorganisms during drying, storageand/or reconstitution. A protective effect during drying mostly comesalong with a stabilizing effect during storage and reconstitution,because when microorganisms are better protected during drying, they areless injured and, as a consequence, more robust during storage andreconstitution.

Numerous ingredients have been suggested in literature as protectiveagents. For example, JP3504365 describes a protective agent forfreeze-drying of bacteria comprising a mixture of at least threeingredients selected from aspartic acid, arginine, glutamic acid,proline, lysine, leucine and methionine. However, freeze-drying anddrying processes using warm gas, such as spray-drying, are verydifferent in nature and expose the biomass to different stress factors.Therefore disclosed protective agents for freeze-drying processes are ofno help to identify protective agents suitable to protect microorganismssuch as bacteria during a drying process using a warm gas, such asspray-drying, and subsequent storage and reconstitution.

Some documents relate specifically to the spray-drying ofmicroorganisms. For example EP1281752 B1 describes a method ofspray-drying microbial cells. Focus is on the size of the powderparticles obtained and on the low temperature used for the dryingprocess. This document also states that a protective agent commonlyknown in the art can be used. It provides numerous examples of potentialprotective agents, namely vitamins such as ascorbic acid; amino acidssuch as glutamine, glutamic acid, cysteine, glycine, phenylalanine,serine or threonine; saccharides or sugar alcohols such as glucose,fructose, sucrose, maltose, mannitol or maltitol; polysaccharides suchas oligosaccharides, cyclodextrin or dextrin; fats such as higher fattyacids obtained from rapeseed, soybean, peanut, etc.; proteins such asthose obtained from cow's milk, soybean, etc. and degraded proteins suchas peptides; inorganic salts such as magnesium sulphate; and others suchas sucrose fatty acid ester, malic acid, nucleic acids, yeast extract,skim milk, peptone, gelatin, tannin, etc. These ingredients can be usedalone or in any combination thereof. This document does not provide anyguidance with regards to selection of particular combinations ofingredients having optimal activity.

Similarly U.S. Pat. No. 6,010,725 relates to spray-drying ofmicroorganisms and their survival. Its teaching is focused on theconditions to be applied during the spray-drying process. It also statesthat a variety of protective agents known in the literature can be usedand recites as examples ascorbic acid, amino acids and their salts suchas lysine, cysteine, glycine and sodium glutamate, proteins or proteinhydrolysates, sugars such as lactose, trehalose, sucrose, dextrin andmaltodextrin, and fats.

Despite the numerous disclosures of abundant lists of ingredients ofpotential use as protective agents, there is a need for furtherrefinement and identification of combinations of ingredients havingoptimized activity to protect microorganisms during drying processesusing warm gas and along subsequent storage and reconstitution.

The present invention advantageously solves the above-mentionedproblems.

SUMMARY

In a first aspect, the invention provides a composition comprising acarrier material comprising a polysaccharide, at least one antioxidantand at least one amino acid selected from lysine, alanine, cysteine andarginine.

In a second aspect, the invention provides for the use of a compositionaccording to the invention for the protection of microorganisms during adrying process using warm gas, during storage and/or duringreconstitution.

In a third aspect, the invention provides a culture powder comprisinglive microorganisms and a matrix comprising a composition according tothe present invention, the matrix:live microorganism dry weight ratiobeing of at least 1.

In a fourth aspect, the invention provides a process for preparing aculture powder comprising

producing a biomass by fermentation with a microorganism;

concentrating the biomass obtained in step a);

conditioning the concentrated biomass with an aqueous solution of acomposition according to the invention; and

drying the conditioned biomass with warm gas to form a powder.

In a fifth aspect, the invention provides a product comprising a culturepowder according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Logarithmic viability loss of Bifidobacterium longum BL999 afterspray-drying and after 2, 4, 6, 8, and 12 weeks storage with Glucidex MDDE 47, lactose, trehalose, maltose or sucrose as protective agent, asmeasured in the pre-screening trial of Example 1.

FIG. 2: Logarithmic viability loss of B. longum BL999 after spray-dryingand after 2, 4, 6 and 8 weeks storage with xylitol, sorbitol, mannitol,lactitol, myo-inositol or glycerol as protective agent, as measured inthe pre-screening trial of Example 1.

FIG. 3: Logarithmic viability loss of B. longum BL999 after spray-dryingand after 2, 4, 6 and 8 weeks storage with sodium glutamate, glutamine,arginine, alanine, lysine or cysteine as protective agent, as measuredin the pre-screening trial of Example 1.

FIG. 4: Logarithmic viability loss of B. longum BL999 after spray-dryingand after 2, 4, 6 and 8 weeks storage with sodium caseinate, wheyprotein isolate, heat denaturated whey protein isolate or gelatin asprotective agent, as measured in the pre-screening trial of Example 1.

FIG. 5: Logarithmic viability loss of B. longum BL999 after spray-dryingand after 2, 4, 6, 8, and 12 weeks storage with gum acacia,

-carrageenan, pectin, guar gum or alginate as protective agent, asmeasured in the pre-screening trial of Example 1.

FIG. 6: Logarithmic viability loss of B. longum BL999 after spray-dryingand after 2, 4, 6, 8, and 12 weeks storage with resistant starch, wheatfibres, raftilose or raftiline as protective agent, as measured in thepre-screening trial of Example 1.

FIG. 7 Logarithmic viability loss of B. longum BL999 after spray-dryingand after 2, 4, 6, 8, and 12 weeks storage with betain HCL, carnitine orproline as protective agent, as measured in the pre-screening trial ofExample 1.

FIG. 8: Logarithmic viability loss of B. longum BL999 after spray-dryingand after 2, and 3 weeks storage with lecithin, a mixture of lecithinand maize oil, a mixture of lecithin and sunflower oil or a mixture oflecithin and soybean oil as protective agent, as measured in thepre-screening trial of Example 1.

FIG. 9: Logarithmic viability loss of B. longum BL999 after spray-dryingand after 2, 4, 6, 8 and 12 weeks storage with Variants A to D and theReference as protective agent, as measured in the trial withcombinations of ingredients in Example 1.

FIG. 10: Logarithmic viability loss of B. longum BL999 afterspray-drying and after 2, 4, 6, 8 and 12 weeks storage with Variants Eto H and the Reference as protective agent, as measured in the trialwith combinations of ingredients in Example 1.

FIG. 11: Logarithmic viability loss of B. longum BL999 after 8 weeksstorage after air convective drying in Samples 1, 5, 6 and 7 (allcomprising L-lysine, either alone or in pair with one other amino acidselected from L-cysteine, L-alanine and L-arginine), as measured inExample 2.

FIG. 12: Logarithmic viability loss of B. longum BL999 after 8 weeksstorage after air convective drying in Samples 2, 5, 8 and 9 (allcomprising L-arginine, either alone or in pair with one other amino acidselected from L-cysteine, L-alanine and L-lysine), as measured inExample 2.

FIG. 13: Logarithmic viability loss of B. longum BL999 after 8 weeksstorage after air convective drying in Samples 3, 6, 8 and 10 (allcomprising L-cysteine, either alone or in pair with one other amino acidselected from L-arginine, L-alanine and L-lysine), as measured inExample 2.

FIG. 14: Logarithmic viability loss of B. longum BL999 after 8 weeksstorage after air convective drying in Samples 4, 7, 9 and 10 (allcomprising L-alanine, either alone or in pair with one other amino acidselected from L-cysteine, L-arginine and L-lysine), as measured inExample 2.

FIG. 15: Cell counts of Streptococcus thermophilus ST496 after 4, 8 and12 weeks storage in Variants I and J and in the Reference, as measuredin Example 3.

FIG. 16: Cell counts of Lactobacillus rhamnosus LPR after 4, 8 and 12weeks storage in Variants K and L and in the Reference, as measured inExample 4.

FIG. 17: Cell counts of Bifidobacterium lactis BL818 after 15, 30, 60,90 and 180 days storage in Variant M and in the Reference, as measuredin Example 5.

DETAILED DESCRIPTION Definitions

“Protective agent”: For the purpose of the present invention, a“protective agent” shall be understood as a composition which iseffective to improve the viability of a live microorganism duringdrying, storage and or reconstitution.

“Drying” or “drying using warm gas”: For the purpose of the presentinvention “drying” and “drying using warm gas” are used indistinctivelyand refer to any process leading to dehydration of a concentrate into apowder under the effect of a gas having a temperature higher than roomtemperature. The gas may be under pressure or at atmospheric pressure.Such processes are as for example spray-drying, atmospheric drying, airconvective drying or fluid bed drying. In a most preferred embodiment,drying refers to spray-drying.

“Reconstitution”: For the purpose of the present invention,reconstitution refers to dissolution or suspension of a powder in aliquid such as water, a specific reconstitution medium as used inanalytical microbiology, or a drink like milk or juice, for example. Theliquid used for reconstitution may be cold or warm. Preferably it refersto reconstitution with water.

Composition

The composition of the present invention comprises at least one carriermaterial comprising a polysaccharide, at least one antioxidant and atleast one amino acid selected from cysteine, lysine, alanine andarginine.

Preferably the composition of the invention is not a nutritionalcomposition, preferably it is not a complete nutritional composition,i.e. it is not a drink or a food providing balanced nutrients to fulfilthe nutritional needs of a human being or animal consuming it,preferably it is not a nutritional composition constituting a whole mealfor an individual consuming it.

In a preferred embodiment of the invention the composition is aprotective agent. More preferably, it is a protective agent for livemicroorganisms, more preferably for live bacteria, most preferably forlive probiotic bacteria. Even more preferably it is a protective agentfor protecting live microorganisms, such as bacteria, during drying,storage and/or reconstitution. Most preferably, it is a protective agentfor protecting live microorganisms during a drying process using a warmgas, such as spray-drying, during storage and/or during reconstitution.

The carrier comprises a polysaccharide. For example, the carrier can bemaltodextrin, dextrin, cyclodextrin, starch, an oligosaccharide orcellulose. Preferably it is maltodextrin. For example maltodextrinhaving a DE of 5 to 12 can be used.

The carrier forms the bulk of the composition. It is essential to carryout a stable drying process of the composition, for example when thecomposition is dried with a microorganism to form a culture powder. Thecarrier makes it possible for example to carry out spray-drying at lowertemperatures and to avoid stickiness of the powder in the spray-dryingtower.

Preferably, the carrier is present in an amount of 20-60 wt %, morepreferably 30-56 wt % based on the total dry weight of the composition.In an embodiment, the carrier consists of a polysaccharide as definedabove.

The antioxidant can be any kind of antioxidant, such as for examplevitamin C, vitamin E, glutathione, coenzyme Q10, (3-carotene, lycopene,vitamin A or a derivative thereof. Preferably, it is vitamin C or aderivative thereof, most preferably sodium ascorbate. In an embodiment,the antioxidant is not one of the amino acids cysteine, lysine, alanineand arginine, even more preferably, it is not an amino acid. Theantioxidant is an essential component of the composition of theinvention. When admixed with live microorganisms, it contributes totheir protection during drying, storage and/or reconstitution.Preferably the antioxidant such as sodium ascorbate is present in anamount of 13-50 wt %, more prefenutritiorably 20-40 wt %, based on thetotal dry weight of the composition.

The composition further comprises at least one amino acid selected fromcysteine, lysine, alanine and arginine. Preferably, the compositioncomprises a combination of at least two of the amino acids cysteine,lysine, alanine and arginine, more preferably a combination of at leastthree of these amino acids. In a preferred embodiment, the amino acidcombination comprises lysine and/or cysteine. The amino acids arepreferably in the form of their L-enantiomer (L-cysteine, L-lysine,L-alanine and L-arginine).

Preferably the amino acids altogether are present in an amount of 3.5-37wt %, more preferably 3.5-34 wt %, most preferably 8 to 34 wt %, basedon the total dry weight of the composition. Lysine, alanine and arginineare each preferably present in an amount of 0-34 wt %, more preferably8-20 wt %, based on total dry weight of the composition. Cysteine ispreferably present in an amount of 0-12 wt %, more preferably 2-10 wt %,most preferably 2-8 wt %, based on total dry weight of the composition.Using cysteine in lower concentrations than other amino acids isadvantageous in that it brings a sensory benefit. In particular,limiting the concentration of cysteine to at most 10 wt %, preferably atmost 8 wt %, based on the total dry weight of the composition improvesthe taste of the composition, leading to improved acceptability byconsumers.

Particularly preferred amino acid combinations include

cysteine and alanine;

cysteine, lysine and alanine;

lysine and arginine;

cysteine and arginine;

cysteine, lysine and arginine;

lysine, alanine and arginine; and

cysteine, arginine and alanine.

The present inventors have surprisingly identified that cysteine,lysine, arginine and alanine, preferably used in the amounts describedabove have superior protective effects when they are used in a culturepowder, thus leading to reduction of loss of live microorganisms duringdrying, storage and/or reconstitution of the powder.

Example 1 shows in particular that cysteine, lysine, alanine andarginine show a better protective effect compared to all other testedindividual ingredients, including other amino acids such as proline,sodium glutamate or glutamine, as well as various other kinds ofsubstances and mixtures such as sugars, sugar alcohols, proteins, gums,fibres and emulsions. Tests with combinations of ingredients in Example1 also show that best results are obtained when these amino acids areused.

A synergistic effect is observed when amino acids are combined in thecomposition, in particular when cysteine and/or lysine is present.Indeed, as shown in Example 2, compositions comprising two amino acids,including lysine and/or cysteine, show better protective effect for livemicroorganisms during drying, storage and/or reconstitution thancompositions comprising a single amino acid, at same total amino acidconcentration.

The carrier material, the antioxidant and the at least one amino acidselected from lysine, cysteine, alanine and arginine are the essentialcomponents of the composition. The composition may comprise additionalcomponents. However the present inventors have discovered that improvedprotective effect is achieved when certain types of ingredients are usedin limited concentration or even avoided. Thus it is preferred that theconcentration of lactose and inositol in the composition be in the rangeof 0-10 wt %, based on the total dry weight of the composition. Morepreferably the composition is free of lactose and inositol. The presentinventors have surprisingly discovered that such compositions with lowlactose and inositol content has superior protective effect for livemicroorganisms during drying, storage and/or reconstitution. For thepurpose of the present invention, “inositol” is meant as being in theform of any of its stereoisomers (myo-inositol, scyllo-inositol,muco-inositol, D-chiro-inositol, neo-inositol, L-chiro-inositol,alto-inositol, epi-inositol and cis-inositol). The most common form ofinositol is myo-inositol and therefore in an embodiment inositol isdefined as myo-inositol.

In a preferred embodiment, the total concentration of all the essentialingredients (carrier, antioxidant and any one or more of the amino acidslysine, cysteine, alanine and/or arginine) is comprised between 80 and100 wt % and the total concentration of additional ingredients iscomprised between 0 and 20 wt %, these percentages being defined basedon the total dry weight of the composition.

In a most preferred embodiment of the invention, the compositionconsists essentially of at least one carrier, at least one antioxidantand at least one amino acid selected from lysine, cysteine, alanine andarginine, as defined in any of the above embodiments, preferably in theamounts indicated. More preferably the composition consists of at leastone carrier, at least one antioxidant and at least one amino acidselected from lysine, cysteine, alanine and arginine, as defined in anyof the above embodiments, preferably in the amounts indicated. Mostpreferably, it consists of maltodextrin, sodium ascorbate and at leastone amino acid selected from lysine, cysteine, alanine and arginine, asdefined in any of the above embodiments, preferably in the amountsindicated.

Use of the Composition

The composition according to any of the above-described embodiments canadvantageously be used for the protection of live microorganisms duringa drying process, such as spray-drying, storage and/or reconstitution.Preferably storage and/or reconstitution are subsequent to a dryingprocess, preferably a spray-drying process. Preferably, the protectiveagent renders it possible to limit the loss of live microorganismsduring a drying process and subsequent storage for a 12 weeks period at37° C. to maximum 1.5 log, more preferably less than 1 log and toachieve a shelf life of 12 months at room temperature in a product inpowder form.

The absolute log viability loss may vary depending on the microbialstrain, the storage temperature, the storage time and the water activityof the medium during storage, such as for example the product in whichthe microorganism is incorporated. Irrespective of the absolute logviability loss measured for one particular strain, the protective effectachieved by the present invention involves a reduction of the viabilitylog loss of that microbial strain compared to the same microbial straindried, stored and/or reconstituted without the composition of theinvention, and preferably also compared to the same microbial straindried, stored and/or reconstituted with a protective agent consisting ofthe carrier and the antioxidant alone (i.e. without amino acid).

The live microorganism can be any kind of live microorganism. Preferablythe microorganisms are bacteria, more preferably beneficial bacteria,such as for example probiotic bacteria. Probiotic bacteria are definedas bacterial cell preparations with a beneficial effect on the health orwell-being of the host (Salminen S, Ouwehand A. Benno Y. et al.“Probiotics: how should they be defined” Trends Food Sci. Technol.1999:10 107-10).

Microorganisms are considered as “live” when they are able to multiplyunder controlled culture conditions and form colonies or suspensions orwhen the microorganism metabolic activity and/or membrane integrity canbe established using methods known to the person skilled in the art,such as for example flow cytometry.

The examples provided in the present application show that thecomposition of the present invention is successful in protecting diversemicrobial strains during drying and subsequent storage andreconstitution. The effect is therefore not strain-specific and can beapplied to a wide range of microbial strains.

Examples of bacteria which can be protected by the composition of thepresent invention include bifidobacteria, lactobacilli, lactococci,enterococci, streptococci, Leuconostoc, Escherichia, propionibacteria,or combinations thereof, preferably Bifidobacterium longum,Bifidobacterium lactis, Bifidobacterium animalis, Bifidobacterium breve,Bifidobacterium infantis, Bifidobacterium adolescentis, Lactobacillusacidophilus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillussalivarius, Lactobacillus rhamnosus, Lactobacillus johnsonii,Lactobacillus plantarum, Lactobacillus fermentum, Lactococcus lactis,Streptococcus thermophilus, Lactococcus diacetylactis, Lactococcuscremoris, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillusdelbrueckii subsp. Lactis, Lactobacillus helveticus, Escherichia coli,Enterococcus faecium, Leuconostoc pseudomesenteroides, Bifidobacteriumbifidum, Lactobacillus gasseri, Lactobacillus sakei, Streptococcussalivarius, and/or mixtures thereof, as well as any of their subspecies.

Examples of bacterial strains that can efficiently be protected IncludeBifidobacterium longum BL999 (ATCC BAA-999), Bifidobacterium longum(CNCM I-2618), Bifidobacterium breve (CNCM I-3865), Bifidobacteriumlactis BL818 (CNCM I-3446), Lactobacillus johnsonii La1 (CNCM I-1225),Lactobacillus paracasei (CNCM I-2116), Lactobacillus rhamnosus LPR(CGMCC 1.3724), Streptococcus thermophilus (CNCM I-1422), Streptococcusthermophilus ST496 (CNCM I-4153), Lactobacillus casei (CNCM I-1518),Lactobacillus casei (ACA-DC 6002), Escherichia coli Nissle,Lactobacillus bulgaricus (CNCM I-1198), Lactococcus lactis (CNCMI-4154), or combinations thereof.

In an embodiment the microorganism is selected from Bifidobacteriumlongum BL999 (ATCC BAA-999), Streptococcus thermophilus ST496 (CNCMI-3915), Lactobacillus rhamnosus LPR (CGMCC 1.3724) and Bifidobacteriumlactis BL818 (CNCM I-3446).

Culture Powder

The present inventors have now developed a culture powder with improvedstability during storage and/or reconstitution. The culture powdercomprises live microorganisms and a matrix comprising a composition asdefined in any of the embodiments described above under the title“Composition”. Preferably the culture powder comprises a matrix and livemicroorganisms, wherein the matrix consists of a composition of theinvention, such as described above under the title “Composition”. Mostpreferably, the culture powder consists of a matrix and of livemicroorganism, wherein the matrix comprises, or even more preferablyconsists of, a composition of the invention as described above under thetitle “Composition”.

In order to ensure efficient protection of the live microorganism, thematrix:microorganism weight ratio in the culture powder is preferably ofat least 1. Preferably it is comprised between 1 and 1.2 to achievesuitable protective effect while maintaining an economically effectiveamount of microorganisms.

Preferably the matrix is in a glassy state.

The live microorganisms are as defined above under the title “Use of thecomposition”.

The amount of live microorganisms in the culture powder of the inventioncan be described as colony forming units (CFU). The required amount oflive microorganisms in the culture powder can be very variable anddepends on the microorganism strain and of the intended use of thepowder. For example, in the case of probiotic bacteria the culturepowder may comprise at least 1E+10, more preferably at least 3E+10 andmost preferably at least 1E+11 CFU per gram of culture powder.

The microorganisms in the culture powder of the present invention may bein the form of a dry biomass comprising the microorganisms together withthe solid content of a fermentation medium. Thus the powder may comprisea nitrogen source such as yeast extract, a carbon source such as a sugarand/or various salts suitable for use in a fermentation medium for thegrowth of the microorganism. It may also comprise fermented derivativesof the nitrogen and carbon source, as well as bioactive ingredientsproduced by the microorganisms during fermentation. Such ingredients aretypically present in an amount of 0-60 wt %, preferably 0-50 wt %, basedon the total dry weight of the microorganism component (biomass).Particularly low amounts of such additional ingredients are present aspart of the microorganism fraction when the biomass is washed beforemixing the biomass with the matrix.

The culture powder may comprise other ingredients in addition to themicroorganism and the matrix. Such ingredients may typically beadditional carriers, such as for example polysaccharides, disaccharidesor skim milk powder. Preferably the matrix is in a glassy state, whereasthe additional ingredients are not in a glassy state.

Process for Producing a Culture Powder

The first step of the process of the invention is the production of abiomass by fermentation by microorganisms. Fermentation methods, underaerobic or anaerobic conditions, are commonly known. The skilled personis able to identify suitable components of the fermentation medium andto adjust fermentation conditions based on his general knowledge,depending on the microorganism to be grown. The fermentation mediumtypically comprises

a nitrogen source such as yeast extract,

a carbon source such as a sugar

various growth factors (e.g. minerals, vitamins etc.) required by themicroorganism and

water.

The fermentation is preferably carried out in two steps, a starterfermentation being carried out prior to the main fermentation step. Thefermentation medium can be different for the starter and the mainfermentation or may be identical.

The second step of the process is the concentration of the biomass. Thiscan also be carried out using methods known to the person skilled in theart, such as for example centrifugation or filtration. The total solidcontent of the biomass after concentration is preferably comprisedbetween 10 and 35 wt %, preferably between 14 and 35 wt %, based on thetotal dry weight of the biomass (i.e. of the total amount offermentation medium and produced microorganism).

Optionally, the concentration may be preceded or combined with a washingstep to remove residues of the fermentation medium and/or compoundsproduced during fermentation. For example, washing may be performed byconcentrating biomass, re-suspending the concentrated biomass in abuffer, such as a phosphate buffer, or a similar composition andre-concentrating the biomass.

The third step of the process is the conditioning of the livemicroorganism. During this operation, a matrix in the form of acomposition of the present invention is contacted with the livemicroorganism. The composition of the present invention is as defined inany of the embodiments described above under the title “Composition”.Preferably, said matrix is a protective agent.

The conditioning step preferably comprises the following sub-steps:

preparing an aqueous solution of the matrix, preferably having a totalsolid (TS) content of 40-65%, more preferably 45-60%.

adding the aqueous solution prepared in step a) to the biomass such asto achieve a matrix concentration of 40-60 wt %, preferably 50-60 wt %,most preferably 55%, based on the total dry weight of matrix and biomass(total solid content);

maintaining the biomass and the matrix into contact for a duration of 20to 150 minutes;

adjusting the pH between 6.5 to 8.5, preferably between 6.8 to 7.2

In one alternative of the process, step c) is carried out before stepd). In another alternative, step d) is carried out before step c).

The desired contact time between the biomass and the protective agentscan be achieved either batch-wise or in a continuous process. When it iscarried out batch-wise, the protective agents and the biomass are mixedin a batch tank and maintained under agitation for the full duration ofthe conditioning time, optionally under cooling conditions, preferablyat T<10° C. to prevent growth of unwanted microbes and sporesgermination. In a continuous process, the desired contact time betweenbiomass and protective agents can be achieved with either a continuousstirred tank reactor with a corresponding average residence time or witha plug flow reactor with a corresponding residence time (similar to aholding tube).

The conditioned biomass is then dried, using any drying method known inthe art using warm gas, such as for example spray-drying, fluid beddrying, air convective drying or atmospheric drying, more preferablyspray-drying. For example, the conditions described in EP0818529, whichis entirely included as reference, can be applied to spray-dryingprocess.

Optionally the dried culture powder may be dry-mixed with additionalingredients. Such ingredients may for example be additional carrierssuch as polysaccharides, as defined above, disaccharides or skim milkpowder.

Product

The present invention also provides a product comprising the culturepowder according to any of the above-described embodiments. The productcan be any type of product in which the culture powder can beincorporated, such as a product in the form of a food or beverageproduct, an animal feed product, a nutritional supplement for human oranimal, a pharmaceutical composition or a cosmetic composition. Theproduct may be intended to be used by the final consumer in solid (suchas powder form) or semi-solid form (such as for example in the form of apaste) or, alternatively, to be reconstituted into a liquid before use.

Food and beverage products include all products intended to be consumedorally by human beings, for the purpose of providing nutrition and/orpleasure. It can for example be a nutritional composition, such as forinfants and/or young children, for a pregnant or lactating woman or awoman desiring to get pregnant, for individuals in need of a specialnutrition due to an adverse health condition or for elderly people. Morepreferably, the nutritional composition is selected from infant formula,infant cereals, follow-up formula, growing-up milks and milk productsfor pregnant and lactating women or for women desiring to get pregnant.Other examples of food and beverage products include dairy products suchas milk products or yogurts, soups, sauces, sweet and savoury snacks,powdered drinks and cereal products.

The product can also be in the form of an animal food product or anutritional supplement for animals. Preferably, the animal is a mammal.Examples of animals include primates (e.g., humans), cows, sheep, goats,horses, dogs, cats, rabbits, rats, mice, fish, birds and the like.

Nutritional supplements are typically present in the form of a powder orof a tablet or capsule. Powder supplements typically encompasssupplements to be dissolved in water or to be sprinkled on food or in abeverage. Such supplements are intended to provide additional nutrientsand/or a health benefit to the subject consuming it, as well as otherbeneficial ingredients, such as beneficial microorganisms, for exampleprobiotic bacteria. A supplement according to the present invention canbe used for providing nutrients and/or a health benefit to human beings,as well as to animals, as defined above. Nutritional supplements includefor example powder supplements to be added to breast milk, for examplefor premature or low birth weight infants. It also includes supplementsfor pregnant or lactating woman or for woman desiring to get pregnant.

Pharmaceutical products include powder, tablet or capsule productsintended to treat of prevent an adverse medical condition in a subjectin need thereof.

Cosmetic compositions are typically intended for an aesthetic effect onthe body and may be for topical use or may be administered by oralroute, in the form of a powder, tablet or capsule.

The product of the present invention preferably comprises livemicroorganisms, preferably beneficial bacteria, such as probioticbacteria, in an amount of at least 5E+06 CFU per gram of product, on adry weight basis.

The ingredients of the matrix can be selected within the above-describedingredients based on the nature of the product, for example based ontaste or regulatory requirements.

The present invention will now be described in further details by theway of the following examples.

Example 1: Protective Agent for Bifidobacterium longum BL999 (ATCCBAA-999)

Sample Preparation

A fermentation medium suitable for the growth of Bifidobacterium longumBL999 was inoculated with 2% frozen-thawed bacteria. The starter wasfermented under the following conditions:

10 hours fermentation time

pH-control with NaOH at pH6.

For the main fermentation, the following conditions were applied:

16 hours fermentation time,

No pH-control

Fermentation broth is covered with CO₂

Starter inoculation: 2%

The biomass was then concentrated by centrifugation to a total solidscontent (TS) of 14% (2.5E+11 CFU/g).

The centrifuged biomass was then conditioned according to the followingprocess. The protective agent of each of the samples was dissolved indemineralised water to a total solids content of 55%. Then 300 g ofcentrifuged biomass were filled into a beaker, which was placed in anice water bath. The total solids content of the biomass was measured andthe protective agent solution was added at a ratio of protective agentsto biomass of 55/45 (based on dry weight). The mixture was gentlyagitated in the ice water bath for one hour. The pH was then adjusted topH7.

The conditioned concentrate was then spray-dried on a lab-scalespray-dryer (Buechi, B-290, Flawil, Switzerland). The air temperaturewas 140° C. and the tower temperature was adjusted by the concentrateflow to T=70° C. During drying, the beaker containing the concentratewas cooled in an ice water bath.

Pre-Screening

In a first round of trials, 38 ingredients were selected as potentialprotective agents. The effect of those ingredients on the stability ofBifidobacterium longum BL999 was tested by spray-drying biomass with aprotective agent comprising maltodextrin DE12, sodium ascorbate and onesingle additional ingredient from the list of 38 compounds to bescreened in the following concentrations:

TABLE 2 Composition of the protective agent for the pre-screeningIngredient Concentration [wt %] Maltodextrin DE12 68.2 Sodium ascorbate13.6 Individual ingredient 18.2 Total 100

A control was prepared with a protective agent consisting of 13.6 wt %sodium ascorbate and 86.4 wt % maltodextrin DE12. This control was usedas a reference to evaluate the effect of the protective agents on thestability of Bifidobacterium longum BL999 during spray-drying, storageand reconstitution.

The 38 culture powders and the control were prepared as described above,the conditioning being carried out with one of the 38 protective agents.

The cell concentration of B. longum in the powder was detected byclassical plating methods. Such classical plating methods are summarizedin the microbiology book: James Monroe Jay, Martin J. Loessner, David A.Golden. 2005. Modern food microbiology. 7th edition, Springer Science,New York, N.Y. The cells were counted in the concentrate before drying,in the culture powder directly after drying and after 2, 3, 4, 6, 8and/or 12 weeks storage in a climatic chamber at T=37° C. abovesaturated potassium acetate solution (a_(w)=0.22). The cell counts afterdrying and after storage were determined after reconstitution of thepowder with a reconstitution medium, as known by the person skilled inthe art, who is well aware of the above-mentioned plating methods.

The following formulae were used for the evaluation of the data:

${{Log}\mspace{14mu} {loss}} = {\log\left( \frac{X_{0}}{X_{t}} \right)}$

where X₀ is the number of micro-organisms at time t=0 and X_(t) thenumber of microorganisms at time t. When the microbiological counts weremeasured in k replicates, X ₀ is the average number of micro-organismsat time t=0 and X _(t) is the average number of micro-organisms at timet, the average log loss at time t is given by:

${{Log}\mspace{14mu} {loss}} = {\log\left( \frac{{\overset{\_}{X}}_{0}}{{\overset{\_}{X}}_{t}} \right)}$

The tested individual ingredients were selected among several groups ofmolecules: sugars, sugar alcohols, amino acids and proteins. All testedingredients within a group were spray-dried on the same day and thereference was spray dried again with each group, also on the same day.

The tested sugars were sucrose, maltose, trehalose, lactose and GlucidexMD DE47. Compared to the reference, sugars did not provide anyprotection against the lethal stresses the bacteria experienced in thespray dryer. While the effect of the sugars on the bacterial survivalduring drying was minor, an impact has been observed on the storagestability. Disaccharides showed the best performance as protectiveagents of which sucrose proved to be the most efficient ingredient.Results are provided in FIG. 1. Sucrose and lactose were selected forthe second round of tests.

The tested sugar alcohols were sorbitol, mannitol, lactitol,myo-inositol, xylitol and glycerol. Spray-drying of the concentrate withsugar alcohols proved difficult due to powder stickiness in the tower.During drying, some of the sugar alcohol showed a light protectiveeffect such as glycerol, mannitol, xylitol and sorbitol. In the storagetest, the powder samples containing glycerol collapsed after a shorttime. The majority of the bacteria of this variant died already aftertwo weeks storage and the storage test was stopped. The viability lossafter 8 weeks was lowest for variants containing sorbitol, mannitol,myo-inositol and lactitol. The results are shown in FIG. 2. For thesecond round of tests, sorbitol, lactitol and myo-inositol wereselected.

The tested amino acids were L-cysteine, L-lysine, L-arginine, L-alanine,L-glutamine and sodium L-glutamate. In contrast to sugars, some aminoacids stabilize the bacteria during drying. While the viability lossduring drying in the reference was 1 log, only 0.2 log was lost for thevariant containing cysteine. Although sodium ascorbate and cysteine areboth known to have antioxidant properties, cysteine surprisingly has theability to protect the microorganism during drying, whereas sodiumascorbate does not show significant effect on the bacterial survivalduring the drying process. Samples containing cysteine, lysine, arginineor alanine showed the lowest losses during storage and reconstitution,among amino acids, but also compared to all other tested ingredients inthis pre-screening phase. These amino acids were selected for the secondround of trials. The results are summarized in FIG. 3.

The tested proteins were gelatin, heat denaturated whey protein isolate(denaturated at T=80° C. for 30 minutes), whey protein isolate andsodium caseinate. None of the proteins improved the stability duringdrying. During storage the heat denaturated whey protein isolate showeda positive effect during the first six weeks, but after 8 weeks theviability loss was as high as for the reference. Due to their weakperformance, no proteins were selected for the second round of trials.The results are summarized in FIG. 4.

All other tested ingredients showed no or very little protectiveactivity and were not selected for the next round of trials:

gums (gum acacia, λ-carrageenan, pectin guar gum and alginate), see FIG.5;

oligosaccharides (resistant starch, wheat fibres, raftilose andraftiline), see FIG. 6.

Other ingredients (betain HCL, carnitine) and the amino acid proline,see FIG. 7.

The pre-screening clearly shows that ingredients mentioned in the priorart as having a potential protective effect for microorganisms duringdrying have very variable efficiency in the protection of livemicroorganism in a drying process using warm gas, such as spray-drying,during storage and/or during reconstitution. The work of the presentinventors has demonstrated the superior efficiency of the amino acidcysteine, lysine, alanine and arginine in combination with a carrier andan antioxidant in the protection of probiotic bacteria duringspray-drying, 12 weeks subsequent storage and reconstitution.

Trials with Combinations of Ingredients

After having selected the most promising ingredients in thepre-screening step, an experimental design was set up to find out thebest protective agents mixes and the optimal concentration profiles.

First, feasibility trials were performed where constraints regarding theprocessing were defined.

The concentration of amino acids altogether had to be limited to a totalof 36.5 wt %, based on the total dry weight of the protective agent.Spray-drying became almost impossible at higher concentrations of aminoacids, due to powder stickiness.

For a stable spray-drying process, a minimum amount of 31 wt % ofmaltodextrin, based on the total dry weight of the protective agent, hadto be incorporated into the protective agent mix.

The content of biomass was fixed to 45 wt % in the final powder and thusthe protective agent amounted to 55 wt %, based on the total dry weightof the biomass and protective agent.

The experimental design comprised 32 variants consisting of variouscombinations of the ingredients identified as promising protectiveagents in the pre-screening trial. Due to capacity of the spray-drier,these variants were divided into groups of 5 variants and a Reference,each group being spray-dried in a single day. As Reference a protectiveagent mix previously identified as having some weak protective effect onmicroorganism during spray-drying was used. The composition of theReference is provided in Table 3 below. In addition to the 5 variants,the Reference was spray-dried on each trial day as well. The Referencewas always spray-dried on a different position in the setup of a trialday to eliminate effects coming from different storage times of thebiomass. The viability loss measurement was measured after all samplesof the group had been spray-dried. Therefore the storage time before themeasurement carried out after spray-drying varied from 0 to 6 hours dueto the time needed to spray dry the 5 variants and the Reference.

TABLE 3 Composition of the Reference Ingredient Concentration [wt %]Maltodextrin 50 Lactose 20 Myo-inositol 9 Sodium glutamate 7 Sodiumascorbate 14 Total 100

All variants were prepared as described for the pre-screening trial,using the combination of ingredients instead of the single ingredient.They were then spray-dried, stored after drying for 12 weeks andanalysed after 2, 4, 6, 8 and 12 weeks, as described above with respectto the pre-screening trials. The same mathematical formulae were usedfor the data analysis. The results of the two groups including the mostpromising variants are provided as FIGS. 9 and 10. The compositions ofthe variants included in these trials are provided in Table 4 below.

TABLE 4 Composition of variants A to H L- cysteine Sodium Myo- L- hydro-L- L- Sodium Variant Maltodextrin ascorbate Sorbitol inositol LactoseSucrose Lactitol Lysine cloride arginine alanine glutamate Ref 50.0 14.00.0 9.0 20.0 0.0 0.0 0.0 0.0 0.0 0.0 7.0 Comparative A 31.8 13.6 9.1 9.10.0 0.0 0.0 0.0 12.2 12.2 12.2 0.0 Invention B 31.8 13.6 9.1 9.1 0.0 0.036.4 0.0 0.0 0.0 0.0 0.0 Comparative C 43.8 36.4 0.0 4.0 0.0 0.0 8.0 2.72.7 2.7 0.0 0.0 Invention D 31.8 34.55 0.0 0.0 0.0 0.0 0.0 11.3 11.3 0.011.3 0.0 Invention E 31.8 34.6 0.0 0.0 11.3 11.3 11.3 0.0 0.0 0.0 0.00.0 Comparative F 31.8 26.6 0.0 0.0 0.0 0.0 20.7 0 6.9 6.9 6.9 0.0Invention G 37.6 36.4 9.1 9.1 0.0 3.8 0.0 1.3 1.3 0.0 1.3 0.0 InventionH 31.8 29.6 0.0 12.9 25.6 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Comparative

The results depicted in FIG. 9 show that Variant B and the Reference,which do not contain any of the amino acids lysine, cysteine, arginineand/or alanine required in the present invention have a much weakerprotective effect, as the logarithmic viability loss for these twosamples is much higher than for the variants of the invention. The bestperforming variant in this design of experiment is Variant D, havingthree amino acids and being free of other ingredients (i.e. it consistsof sodium glutamate, maltodextrin, and the amino acids lysine, cysteineand alanine). On FIG. 10, variants F and G according to the inventionalso show much better protective effect than Variants E and H (having noamino acid).

This example shows that the protective effect is provided withprotective agents comprising diverse mixtures of the amino acidscysteine, lysine, alanine and arginine, in a broad range of totalconcentrations, ranging from 3.8 to 36.5 wt %, based on the total dryweight of the protective agent.

Example 2: Comparison of Diverse Amino Acids Ratios

Samples of culture powder were prepared comprising Bifidobacteriumlongum BL999 (ATCC BAA-999) and a matrix comprising maltodextrin, sodiumascorbate and the amino acids cysteine, lysine, alanine and argininealone or in combinations by pairs, as described in Table 5 below.

TABLE 5 Amino acid composition in the matrix of tested samples Sampleno. Lysine (%) Arginine (%) Cysteine (%) Alanine (%) 1 34 0 0 0 2 0 34 00 3 0 0 34 0 4 0 0 0 34 5 17 17 0 0 6 17 0 17 0 7 17 0 0 17 8 0 17 17 09 0 17 0 17 10 0 0 17 17

All percentages are defined by weight, relative to the total weight ofthe matrix.

The samples were prepared with 55% matrix and 45% of Bifidobacteriumlongum BL999 (ATCC BAA-999), the percentage being defined by weight,based on the total dry weight of the culture powder. The matrixconsisted of 33% maltodextrin DEX, 33% sodium ascorbate and 34% of aminoacid composition as described in Table 5, the percentages being definedby weight relative to the total weight of the matrix.

The samples were prepared as follows. B. longum BL999 was grown for 16 hat 37° C. in a 7-L fermenter (newMBR, CH-Zürich) under CO2-headspaceatmosphere using the same fermentation medium as in Example 1. The pH ofthe fermentation was not controlled. Cell suspension collected from thefermenter was centrifuged at 4500 rpm for 20 min at 5° C. (Sorvall RC3CPlus). After discarding the supernatant, the biomass was stored at 4° C.prior to mixing with the protective matrix. The pH of the concentratewas adjusted to 7.0 by drop-wise addition of 30% NaOH before drying withanalytical convective dryer (ACD) at lab scale (built by IVV-FraunhoferInstitut, DE-Freising). Relevant drying parameters such as air humidity,air temperature and air flow rate were adjusted as follows:

Air flow: 40 m³/h

Temperature of the drying air: 60° C.

Relative humidity of drying air 8%

Duration 660 s

The log loss was analyzed for each sample as described in Example 1,after drying, and after 8 weeks storage above saturated sodium acetatesolution (a_(w)=0.22) in a climatic chamber at T=37° C. The cellconcentration of B. longum BL999 was measured before drying and after 8weeks storage using the method described in Example 1. The log loss wasalso calculated using the equations described in Example 1.

The results are provided in FIGS. 11 to 14. These figures show thatsamples in which a pair of amino acids was used in the protective agentexhibited reduced log viability loss compared to samples where only oneamino acid was used. The only exception is the sample produced with acombination of the two amino acids alanine and arginine, which showedincreased viability loss compared to arginine or alanine alone. Eventhough the effect of the combination of alanine and arginine proved tobe lower than that of the individual ingredients, the results providedevidence of significant protective effect of alanine and arginine assingle ingredients or combined in a pair.

These results provide evidence of a synergy between the amino acids whenused in pairs, in particular when cysteine and/or lysine is present,compared to the use as single amino acid, because at sameconcentrations, pairs of amino acids provide better stabilization thansingle amino acids.

Example 3: Protective Agent for Streptococcus thermophilus ST496 (CNCMI-3915)

A comparative experiment has been performed to evaluate the performanceof a composition according to the invention for the protection of themicroorganism Streptococcus thermophilus ST496 (CNCM I-3915) duringstorage and reconstitution.

A Reference and two samples (Variants I and J) were prepared. AsReference a protective agent mix previously identified as having someweak protective effect on microorganism during spray-drying was used.The composition of the Reference is provided in Table 3 above. Theprotective agent composition used in each test sample is given in Table6 below. For all samples the matrix concentration was of 55 wt % and themicroorganism concentration was of 45 wt %, based on total solids.

TABLE 6 Composition of samples I and J Ingredient Variant I Variant JMaltodextrin DE6 55.00 50.00 Sodium ascorbate 22.32 25.00 L-Lysine HCL9.34 10.30 L-Cysteine HCL monohydrate 4.00 4.40 L-Alanine 9.34 10.30Total 100.00 100.00

The Reference and test samples were all prepared using the same method,as follows. S. thermophilus was produced in 3000 L bioreactor at 40° C.for 9 h at pH constant after inoculation of a suitable medium containingnitrogen source, carbon source and trace elements at 2% with the starterculture. Biomass at end of fermentation was concentrated bycentrifugation and conditioned with the matrix for 1 h. Biomass andmatrix after conditioning and pH adjustment at 7 were spray-dried atlarge scale using a spray drying tower size wise close to an industrialscale drying tower.

After spray-drying, the Reference and test samples were subjected to anaccelerated storage test. The culture powder was mixed with skim milkpowder at a ratio of 1 to 100, after spray-drying of the culture powder.Then this mixture was stored at 37° C. and a_(w)=0.25 for 12 weeks. Thecell counts were measured in the Reference and in the test samplesimmediately after spray-drying and after 4, 8 and 12 weeks storage. Thecell counts were measured as described in Example 1.

The results are shown in FIG. 15. It can be seen from the graph that thetotal microorganism cell counts are significantly higher after 12 weeksstorage and reconstitution in Variants I and J (according to theinvention), than in the Reference. This shows that the composition ofthe invention is effective in protecting live Streptococcus thermophilusST456 during storage and reconstitution after spray-drying. Whencombining this observation with what is obtained in the other examples,it shows that the protective effect of the composition of the inventionis not strain specific and is effective on a large variety ofmicroorganism strains. In addition, the effect is achieved withdifferent concentrations of the ingredients of the protectivecomposition. This example also shows that the protective effect isobtained when the culture powder is produced at large scale.

Example 4: Protective Agent for Lactobacillus rhamnosus LPR (Depositedas CGMCC 1.3724)

A comparative experiment has been performed to evaluate the performanceof a composition according to the invention for the protection of themicroorganism Lactobacillus rhamnosus LPR (deposited as CGMCC 1.3724)during spray-drying, storage and reconstitution.

A Reference and two samples (Variants K and L) were prepared. AsReference a protective agent mix previously identified as having someweak protective effect on microorganism during spray-drying was used.The Reference composition is provided in Table 3 above. The protectiveagent composition used in each test sample is given in Table 7 below.For all samples the matrix concentration was of 55 wt % and themicroorganism concentration was of 45 wt %, based on total solids.

TABLE 7 Composition of samples K and L Ingredient Variant I Variant JMaltodextrin DE6 60.00 55.00 Sodium ascorbate 19.85 22.32 L-Lysine HCL8.30 9.34 L-Cysteine HCL monohydrate 3.55 4.00 L-Alanine 8.30 9.34 Total100.00 100.00

The Reference and test samples were all prepared using the same method,as follows.

L. rhamnosus was produced in 3000 L bioreactor at 37° C. for 16 h at pHconstant after inoculation of a suitable medium containing nitrogensource, carbon source and trace elements was used to obtain high yieldat 4% with the starter culture. Biomass at end of fermentation wasconcentrated by centrifugation and conditioned with the matrix for 1 h.Biomass and matrix after conditioning and pH adjustment at 7 werespray-dried at large scale using a spray drying tower size wise close toindustrial scale drying tower.

After spray-drying, the Reference and test samples were subjected to anaccelerated storage test. The culture powder was mixed with skim milkpowder at a ratio of 1 to 100, after spray-drying of the culture powder.Then this mixture was stored at 37° C. and a_(w)=0.24 for 12 weeks. Thecell counts were measured in the Reference and in the test samplesimmediately after spray-drying and after 4, 8 and 12 weeks storage. Thecell counts were measured as described in Example 1.

The results are shown in FIG. 16. It can be seen from the graph that thetotal microorganism cell counts are significantly higher after 12 weeksstorage and reconstitution in Variants K and L (according to theinvention), than in the Reference. This shows that the composition ofthe invention is effective in protecting live Lactobacillus rhamnosusLPR during storage and reconstitution after spray-drying. When combiningthis observation with what is obtained in the other examples, it showsthat the protective effect of the composition of the invention is notstrain specific and is effective on a large variety of microorganismstrains. In addition, the effect is achieved with differentconcentrations of the ingredients of the protective composition. Thisexample also shows that the protective effect is obtained when theculture powder is produced at large scale.

Example 5: Protective Agent for Bifidobacterium lactis BL818 (Depositedas CNCM I-3446)

A comparative experiment has been performed to evaluate the performanceof a composition according to the invention for the protection of themicroorganism Bifidobacterium lactis BL818 (deposited as CNCM I-3446)during spray-drying, storage and reconstitution.

A Reference and one test sample (Variant M) were prepared. As Referencea protective agent mix previously identified as having some weakprotective effect on microorganism during spray-drying was used. Thecomposition of the Reference is provided in Table 3 above. Theprotective agent composition used in the test sample is given in Table 8below. For all samples the matrix concentration was of 55 wt % and themicroorganism concentration was of 45 wt %, based on total solids.

TABLE 8 Composition of samples M Ingredient Variant M Maltodextrin DE652.00 Sodium ascorbate 23.80 L-Lysine HCL 10.00 L-Cysteine HCL 4.20L-Alanine 10.00 Total 100.00

The Reference and the test sample were prepared using the same method.

After spray-drying, the Reference and the test sample were subjected toan accelerated storage test at 37° C. and a_(w)=0.17 for 180 days. Thecell counts were measured in the Reference and in the test sampleimmediately after spray-drying and after 15, 30, 60, 90 and 180 daysstorage. The cell counts were measured as described in Example 1.

The results are shown in FIG. 17. It can be seen from the graph that thetotal microorganism cell count is significantly higher after 180 daysstorage and reconstitution in Variants M (according to the invention),than in the Reference. This shows that the composition of the inventionis effective in protecting live Bifidobacterium lactis BL818 duringstorage and reconstitution after spray-drying. The log viability loss(calculated as described in Example 1) is of only 0.24 after 180 days inthe sample of the invention, showing a very efficient protective effect.

When combining this observation with what is obtained in the otherexamples, this example shows that the protective effect of thecomposition of the invention is not strain specific and is effective ona large variety of microorganism strains. In addition, the effect isachieved with different concentrations of the ingredients of theprotective composition. This example also shows that the protectiveeffect is obtained when the culture powder is produced at large scale.

The invention is claimed as follows:
 1. A composition comprising acarrier material comprising a polysaccharide, the composition furthercomprising at least one antioxidant and at least one amino acid selectedfrom the group consisting of cysteine, lysine, alanine and arginine. 2.The composition according to claim 1, comprising a combination of atleast two amino acids selected from the group consisting of cysteine,lysine, alanine and arginine.
 3. The composition according to claim 1,wherein the at least one amino acid comprises at least one of cysteineor lysine.
 4. The composition according to claim 1, wherein the carriermaterial is selected from the group consisting of maltodextrin, dextrin,cyclodextrin, starch, an oligosaccharide and cellulose.
 5. Thecomposition according to claim 1, wherein the at least one antioxidantis selected from the group consisting of vitamin C, vitamin E,glutathione, coenzyme Q10, B-carotene, lycopene, vitamin A and aderivative thereof.
 6. The composition according to claim 1, wherein atotal of the at least one amino acid is 3.5 wt. % to 36.5 wt. % of thetotal dry weight of the composition.
 7. The composition according toclaim 1, wherein: the carrier is 20-60 wt. %; the at least oneantioxidant is 13-50 wt. %; any cysteine is no greater than 12 wt. %;any lysine is no greater than 34 wt. %; any arginine is no greater than34 wt. %; and any alanine is no greater than 34 wt. %; all percentagesdefined by weight based on the total dry weight of the composition. 8.The composition according to claim 1 consisting essentially of thepolysaccharide, the at least one antioxidant and the at least one aminoacid selected from the group consisting of cysteine, lysine, alanine andarginine.
 9. A culture powder comprising live microorganisms and amatrix comprising a composition comprising a carrier material comprisinga polysaccharide, the matrix further comprising at least one antioxidantand at least one amino acid selected from the group consisting ofcysteine, lysine, alanine and arginine, a dry weight ratio of thematrix:the live microorganisms is at least
 1. 10. The culture powderaccording to claim 9, wherein the live microorganisms are bacteria. 11.The culture powder according to claim 10, wherein the bacteria isselected from the group consisting of bifidobacteria, lactobacilli,lactococci, enterococci, streptococci, Leuconostoc, Escherichia,propionibacteria, and combinations thereof.
 12. A product comprising aculture powder according to claim 9, wherein the product is selectedfrom the group consisting of a nutritional composition, a food orbeverage product, an animal feed product, a food supplement, apharmaceutical composition and a cosmetic composition.